PROJECT SUMMARY The T cell receptor (TCR)-CD3 complex plays an important role in antigen recognition in cell mediated adaptive immune responses. A fundamental unanswered question in immunology is how ligand engagement of TCR mediates critical information transfer from the antigen recognition site in the external environment to the intracellular compartment via the CD3 signaling complex, resulting in signal initiation. A complete understanding the molecular organization and function of the TCR-CD3 signaling complex has wide significance for development of new strategies for immune therapeutic intervention focusing on the very earliest stages of immune activation. The overall goal of this project is to understand how antigen recognition leads to molecular changes in the extracellular TCR-CD3 complex that further propagates molecular changes in intracellular immunoreceptor tyrosine-based activation motifs (ITAM) to result in functional outcomes. Our hypothesis is that force-induced or spontaneous reorientations and/or conformational changes in the extracellular TCR-CD3 complex and intracellular ITAMs upon agonist ligand binding are necessary for sufficient binding of protein tyrosine kinases (Lck, ZAP-70) to initiate proximal signaling events. Based on our recently published work that generated a structural model of the extracellular assembly of the TCR-CD3 complex and our preliminary data demonstrating that there is feedback mechanism through Lck mediated CD8 binding to agonist pMHC that prolongs TCR-pMHC catch-bonds we propose the following three aims to test our hypothesis. In the first aim, we will use paramagnetic relaxation enhancement, biomembrane force probe (BFP) assay and photo-crosslinking to determine how extracellular arrangements in the TCR-CD3 complex influence intracellular signaling. In the second aim, we will use NMR chemical shift perturbation analysis and BFP to study the interaction of intracellular CD3 with Lck/ZAP-70 to determine how structural changes in CD3 cytoplasmic domains influence protein tyrosine kinases interaction. In the third aim, we will employ BFP combined with concurrent FRET based imaging of proximal signaling components to determine how changes in TCR-CD3 extracellular interaction influence CD3-Lck /ZAP-70 interactions, TCR-pMHC binding and TCR- pMHC-CD8 interactions. By undertaking these aims we expect to determine the mechanistic basis of the signal transduction process through the TCR-CD3 complex during T cell recognition. Providing a detailed understanding of the involvement of force and specifics of these molecular interactions will allow for a more complete understanding of the molecular basis of signal transduction process during T cell antigen recognition. The relevance of this work to public health is in the potential to improve the response rate and overall outcomes of immunotherapies. Potential treatment strategies could include the use of antibodies, modified T cells, chimeric antigen receptors or small molecules which would allow for manipulation of immune responses to cancer, infections, inflammation and autoimmunity, which remains a significant problem in human health.